Method and device for nucleic acid sequencing

ABSTRACT

A method for sequencing a nucleic acid strand, comprising the steps of: providing a solution containing truncated strands having lengths different from one another terminating with a respective dideoxynucleotide from among ddATP, ddTTP, ddGTP, and ddCTP; functionalizing first masses by a donor molecule and second masses by an acceptor molecule such as to generate a light emission when they come into mutual contact; coupling a first mass to a first end of each truncated strand; coupling the second masses to a respective terminal dideoxynucleotide of each strand; applying an AC electrical field having variable frequencies that are such as to generate, on each second mass, a net movement directed towards the first mass; acquiring a plurality of light radiations for each frequency value; and associating each light radiation acquired to a respective dideoxynucleotide and, thus, to a respective nucleotide base.

BACKGROUND Technical Field

The present disclosure relates to a method and a device for nucleic acidsequencing.

Description of the Related Art

Known in the art are methods for DNA sequencing. Sequencing isfundamental for characterizing a macromolecule, for example in order todetermine the order of the amino acids of a protein or the sequence ofbases of a nucleic acid. Sequencing of an entire genome may enableprediction of the sequence of all the proteins that this is potentiallyable to produce.

A method for DNA sequencing known in the prior art envisages the use ofdideoxynucleotide terminators and is known as the “Sanger method”. TheSanger method consists of three steps: preparation of the sample,sequencing reaction, and electrophoresis. In the first step ofpreparation of the sample, the DNA strand that is to be sequenced issubjected to PCR (Polymerase Chain Reaction) in order to amplify it,i.e., obtain a plurality of identical copies thereof. Other techniquessuch as recombinant DNA may also be used. In the second, sequencingreaction step, the biological sample is subjected to denaturing, primerannealing, copy of the strand, and termination. With denaturing, the DNAis separated into individual strands. During primer annealing, a primeris added to the end 3′ of one of the two strands. The primer issynthesized artificially and appropriately for the DNA sequence to besequenced.

Four mixtures are then prepared, one for each base, added to which isDNA polymerase (step of copy of the strand), the four nucleotides (dATP,dCTP, dGTP, dTTP), and an amount of a dideoxynucleotide triphosphate(ddNTP, for example, ddATP), i.e., a nucleotide without the —OH group inposition 3′ of the sugar.

The above may, however, be added by the DNA polymerase to a DNA strandbeing synthesized via formation of a phosphodiester bond between its5′-phosphate and 3′-OH of the previous residue. However, since ddNTPslack the —OH group in position 3′, the subsequent nucleotide may not bebound as occurs in natural DNA replication. For this reason, thesynthesis stops at the position in which a ddNTP has been incorporatedat the growing end of a DNA strand (termination step).

The new strands, each of which terminates with a ddNTP, for instance addATP in the example considered, have lengths that are different fromone another. Once DNA polymerase encounters a T base on the templatestrand, it may add a dATP or a ddATP. If a dATP is added, growth of thestrand continues, whereas if a ddATP is added, growth of the strandstops. This process is carried out for all four nucleotides.

Then, the strands thus synthesized are denatured and separated from thetemplate strands. At this point, the preparation is ready for theelectrophoresis step.

Electrophoresis is an electrokinetic process in which charged moleculesand particles, under the influence of an electrical field, migrate inthe direction of a pole that has opposite charge from that of thecharged molecules. Owing to the presence of the phosphate groups, theDNA molecules are negatively charged and will thus migrate towards thepositive pole (anode) if subjected to an electrical field, with a ratethat depends also upon their length, as well as upon the fieldintensity.

Introduction of capillary electrophoresis for separation of markedfragments has enabled a considerable increase in the processing rate.There have further been developed automatic sequencers that are able tocarry out multiple electrophoretic runs.

However, the use of the electrophoresis technique renders the Sangermethod impractical to integrate in a portable biomedical device such asone that is obtained using MEMS technology.

New-generation sequencers, which are not based upon the Sanger method,use in-vitro amplification techniques and an array system forsimultaneous sequencing of millions of DNA fragments. These improvementshave enabled new platforms to reduce drastically the times and costsinvolved even though they require a demanding post-processing step andpresent limits of precision above all in counting the occurrences ofrepeated sequences. Further, these techniques are excessive in terms ofcost and complexity for cases where it is necessary to focus sequencingon small selected parts of the genome.

Alongside the sequencing systems on a large scale, there is in factcurrently felt the need for a method for target sequencing, typically ofa single isolated gene, of which it is desired to know the exactsequence of the bases and possible variants. Such a method, possiblyintegrated with a PCR amplification system, would prove useful in thecase where (for example, during a diagnostic examination) there isidentified the presence of a particular gene, of which it is necessaryto know the exact sequence (variant).

BRIEF SUMMARY

According to the present disclosure, a method and a device for nucleicacid sequencing are thus provided, as defined in the annexed claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

For a better understanding of the present disclosure, preferredembodiments thereof are now described, purely by way of non-limitingexample and with reference to the attached drawings, wherein:

FIG. 1 illustrates a system formed by a single DNA strand coupled tofunctionalized masses, according to an aspect of the present disclosure;

FIG. 2 illustrates the system of FIG. 1 in a reference system centeredon one of the two masses and with forces acting on the other of the twomasses;

FIG. 3 illustrates the system of FIG. 2 in an operating condition ofrelative movement of the two masses;

FIGS. 4A-4E illustrate steps of a control method of the system of FIG.1, according to an aspect of the present disclosure;

FIG. 5 illustrates a device designed to implement the method of FIGS.4A-4E; and

FIG. 6 illustrates the block diagram of a system for DNA amplificationand sequencing, according to an aspect of the present disclosure.

DETAILED DESCRIPTION

According to the present disclosure, one embodiment provides a methodfor sequencing a macromolecule, in particular DNA, based upon the Sangermethod, and in particular including the steps of preparing a sample andsequencing reaction according to the Sanger method without carrying outan electrophoresis step.

The Sanger method is per se known and thus is not described in detailherein, but a possible embodiment thereof is provided by way of example.The steps of preparation of the sample and of sequencing reactionrequire a single-strand DNA template (or template strand), a primer forstarting the polymerization reaction, a DNA polymerase, anddeoxynucleotides and dideoxynucleotides for terminating thepolymerization reaction. The modified nucleotides (ddNTPs) or theprimer, according to the present disclosure, do not have to be marked(either by radioactivity or by fluorescence) in so far aselectrophoresis is not carried out. The DNA sample to be sequenced isdivided into four separate reactions, each of which contains the DNApolymerase and all four deoxyribonucleotides (dATP, dCTP, dGTP, anddTTP). This step may be carried out in a test tube, or else in dedicatedwells provided in an integrated chip, for example of semiconductormaterial. Then added to each of these reactions is just one of the fourdideoxynucleotides (ddATP, ddCTP, ddGTP, and ddTTP) instoichiometrically lower amount in order to enable a lengthening of thestrand sufficient for carrying out analysis. Incorporation of adideoxynucleotide along the growing DNA strand causes terminationthereof before the end of the template DNA sequence is reached. Thisgives rise to a series of DNA fragments of different length interruptedby incorporation of the dideoxynucleotide, which occurs randomly when itis used by the polymerase instead of a deoxynucleotide.

Next, the strands thus synthesized are denatured to obtain separation ofindividual DNA strands from the template strands. A plurality of partialstrands, or DNA fragments, is thus obtained. FIG. 1 represents one ofthese partial strands, designated by the reference number 10. Eachpartial strand typically has a length different from that of the otherpartial strands and terminates with a respective nucleotide base fromamong adenine, thymine, guanine, and cytosine.

Then, according to the present disclosure, each partial strand isassembled to form the structure 1 illustrated in FIG. 1 by binding afirst end 10 a of the partial strand 10 to a first mass 2, and a secondend 10 b of the partial strand 10 to a second mass 4. In the figures,the first and second masses 2, 4 are illustrated, for simplicity ofrepresentation, of a circular or spherical shape, without this implyingthat they must necessarily have this shape.

The second mass 4 is configured to bind exclusively to one of thenucleotide bases, and in particular to the base that terminates thepartial strand 10 prematurely. For this purpose, four different types ofsecond masses 4 are provided, functionalized in an appropriate way forbinding each to a respective dideoxynucleotide that terminates theplurality of partial strands obtained via the aforementioned steps ofthe Sanger method. A first type of second mass 4 is functionalized forbinding to a first dideoxynucleotide (e.g., ddATP), a second type ofsecond mass 4 is functionalized for binding to a seconddideoxynucleotide (e.g., ddTTP), a third type of second mass 4 isfunctionalized for binding to a third dideoxynucleotide (e.g., ddGTP),and a fourth type of second mass 4 is functionalized for binding to afourth dideoxynucleotide (e.g., ddCTP).

The first mass 2 is configured to bind to the first end 10 a of eachstrand 10. For this purpose, the mass may be functionalized for bindingto the PCR-primer sequence.

In solution, the first mass 2 is, as a result of its dimensions and/orother characteristics (e.g., electrical characteristics and/orcharacteristics of hydrodynamic friction), less mobile than the secondmass 4 for functioning as constraint in the oscillating movement of theDNA strand used for sequencing.

In an embodiment of the present disclosure, the first mass 2 is aprotein, responding to the characteristics of the molecules referred toas “drag-tags”. Drag-tag technology is already widely used and studiedas method for free DNA electrophoresis in a liquid in order to determinethe length of various fragments. The fundamental characteristics of thetechnology mentioned are briefly outlined in what follows.

It should be noted that the mobility of DNA, once a certain criticallength of the strand has been exceeded, no longer depends upon thelength itself, in so far as the ratio between the electrostatic pullingforce (which depends linearly upon the charge at constant density, andthus upon the length of the strand) and the force of hydrodynamicfriction (which also depends linearly upon the length of the strand) isconstant. By attaching the strand with a molecule with a highcoefficient of hydrodynamic friction (i.e., the drag-tag), the frictionitself depends above all upon said additional molecule, whereas thepulling force continues to depend upon the length of the strand. In thisway, there has been shown separation by electrophoresis in a liquid ofDNA strands of different lengths (within a range that depends upon thecharacteristics of hydrodynamic friction of the drag-tag).

In contrast to the drag-tag technology in the prior art, the presentdisclosure is principally based on a movement of folding of the DNAstrand 10 up to provide mutual contact of the two ends 10 a, 10 b of theDNA strand (the drag-tag is bound to one of the two ends), andconsequent emission of fluorescence. In order to obtain the desiredeffect, it is expedient for the force that guides the oscillatorymovement to be tuned in frequency and/or amplitude with the length ofthe DNA strand to obtain a resonance condition. This enables convenientand precise measurement of the partial lengths, improving the resultsthat may be obtained with electrophoresis in a liquid according to theknown art. Use of drag-tags (including protein drag-tags) in order toincrease the overall hydrodynamic friction is performed, per se,according to the prior art, and thus this aspect is not discussed anyfurther herein.

By way of example, see the paper by R. J. Meagher et al., “Sequencing ofDNA by Free-Solution Capillary Electrophoresis Using a GeneticallyEngineered Protein Polymer Drag-Tag”, Anal. Chem. 2008, 80, pp.2842-2848.

See further the paper by R. D. Haynes et al., Bioconjugate Chem. 2005,16, pp. 929-938.

Further, see the paper by Jong-In Won et al., “Protein polymer drag-tagsfor DNA separations by end-labeled free-solution electrophoresis”,Electrophoresis 2005, 26, pp. 2138-2148.

In an embodiment of the present disclosure, the first mass 2 and thesecond mass 4 are both proteins of the same type as those used fordrag-tags. However, whereas the mass 2 has a drag-tag function proper(i.e., contributing to increasing the overall hydrodynamic friction),the mass 4 preferably exhibits an overall net charge that renders itsensitive to the action of an applied electrical field, as illustratedmore fully in what follows. Since the electrical field is not localizedbut acts also on the DNA strand 10, which in turn presents a non-zeroelectrical charge, it is preferable for the charge of the mass 4 to bemuch higher than the total charge of the strand 10 in order for themovement of the strand 10 itself to be guided by its end. In someembodiments, strand 10 has a maximum length of between 100 and 200bases. Considering an elementary charge C per base, the net charge ofthe mass 4 is higher (indicatively by one order of magnitude) thanapproximately 100e⁻ (e.g., it is approximately 1000e⁻). The charge ispreferably negative to prevent attraction to the DNA strand 10, withconsequent folding on the mass 2 itself.

The mass 4 is bound to the PCR primer, whereas the mass 2 is bound tothe four modified bases. The techniques for functionalizing the primerand the bases, including modified ones, with other molecules are knownin the prior art and thus not described in detail herein.

The first mass 2 is further functionalized with a donor molecule 6,whereas the second mass 4 is functionalized with an acceptor molecule 8.The donor molecule 6 and the acceptor molecule 8 are chosen so that,when they are brought at a distance from one another shorter than aminimum distance, there is a transfer of energy from the donor molecule6 to the acceptor molecule 8, resulting in emission of light.

Donor and acceptor molecules that may be used include 3′-fluorescein and5′-LC Red 640, respectively.

In the condition of FIG. 1, when the first and second masses 2, 4 are ata distance from one another and the partial strand 10 is splayed outcompletely or in part, there is no transfer of energy from the donormolecule 6 to the acceptor molecule 8. On the other hand, in conditionswhere the first mass 2 and the second mass 4 are apart from one anotherat a distance shorter than the minimum distance, energy transfer occurs,which cause emission of light that may be detected. See e.g., FIG. 4C.In one embodiment, the donor molecule 6 and the acceptor molecule 8 arechosen so that said minimum distance is less than a few nanometers,preferably less than 1 nm.

According to an aspect of the present disclosure, the structure 1 ofFIG. 1 is subjected to a variable electrical field (e.g., sinusoidal) inorder to vary in a controlled way the distance between the first andsecond masses 2, 4. The electrical field applied acts on the first andsecond masses 2, 4 and, as a result of the difference in size, and/orweight, and/or hydrodynamic friction between the two masses 2, 4,generates a relative movement of the second mass 4 with respect to thefirst mass 2.

Preferably, in an initial step of the method, a DC component is appliedto generate a force that causes the first mass 2 to pull away from thesecond mass 4, thus stretching the strand 10. Said DC component mayfurther cause a movement in space of the entire system 1, i.e., of boththe masses 2, 4 and the strand 10, in an undesired way. For thispurpose, it is advisable to reverse periodically the direction of the DCfield for generating a substantially zero net displacement of the system1. The DC electrical field may, for this purpose, be a signal thatassumes periodically values K, 0, −K, 0, etc., where |K| is a valuegreater than zero. In one embodiment, the periods of high or low signal“K” have the same temporal duration, as the periods of zero signal “0”.The average is a zero signal. It is possible to use, for example, asquare-wave signal with zero average.

With reference to the model represented schematically in FIG. 2, asystem of co-ordinates X, Y, and Z is considered having origin O at thecenter of mass of the first mass 2.

After the DC electrical field has been applied and the DNA strand 10 hasbeen splayed out, the DC field is turned off, and an AC electrical fieldis applied having a first component F_(X) along the direction X and asecond component F_(Y) along the direction Y orthogonal to the directionX. A force component oriented along a third direction Z, orthogonal to Xand Y, may be of zero value, thus allowing the second mass 4 to movealong Z with Brownian motion. Brownian motion along Z is a motion withzero average and thus does not represent a net component of movement ofthe system as a whole.

The component F_(X) and the component F_(Y) are, in this example,quadrature sinusoidal signals with a frequency ratio equal to 2 or amultiple of 2, and specifically

F _(X) =A·cos(ωt)

F _(Y) =B·sin(Mωt)

where: A and B are the respective values of signal amplitude and are,for example, chosen with a value comprised between −10 pN and +10 pN(the sign depends upon the direction of the force); w is the angularfrequency (equal to 2πf, where f is the frequency, for example rangingbetween 100 MHz and 3 GHz); M is the ratio, chosen, as has been said,equal to 2 or a multiple of 2; and t is the time variable, measured inseconds.

For a nucleic acid strand of 40 bases, it has been found that afrequency of approximately 1 GHz yields good results, with an estimatedsensitivity (frequency variation) of approximately 75 MHz per base.

However, it is possible to scale, also by several orders of magnitude,the frequencies and the forces, in a way that is interdependent and, inturn, depends upon the overall charge of the mass 4.

In the condition of FIG. 2, the value of amplitude B is greater thanzero, whereas the value of amplitude A is smaller than zero in so faras, at a first instant of time, the force F_(X) is applied along thenegative direction of the axis X. The present applicant has in factfound that application of a positive force F_(X), at an instantimmediately subsequent to the step of stretching of the strand 10 by theDC field, may cause the strand 10 to break.

With reference to FIG. 3, the force F_(X) counters an elastic forceF_(el) proper to the strand 10, which tends to cause the strand 10 toroll up in order to reach a condition of maximum entropy, whereas theforce F_(Y) generates a movement that displaces the second mass 4 alonga semi-curvilinear path around the first mass 2, imparting on the secondmass 4 an oscillatory motion that displaces the strand 10 from theoriginal position (represented in FIG. 3 by a dashed line).

With reference to FIGS. 4A-4E, illustrated graphically therein is theeffect of application of the AC electrical field that generates theforce F_(X) and the force F_(Y).

At time t=t₀ (FIG. 4A), the strand 10 is assumed as being completelysplayed out, as a result of the component of DC field applied at animmediately previous instant. The force F_(X) has, in t=t₀, a maximumvalue, i.e., equal to |A|, and a direction such as to move the first andsecond masses 2, 4 away from one another (e.g., F_(X)=−10 pN).

The first mass 2 is illustrated coupled to a donor molecule 6 and thesecond mass 3 is illustrated coupled to an acceptor molecule 8.

Then (FIG. 4B), the force F_(X) decreases according to a sinusoidalpattern until it reaches a zero value (point of change of sign of theforce F_(X)). In this situation, the strand 10 undergoes first theaction of the elastic force F_(el), and then the action of the forceF_(X), which favor an “arching” of the strand 10, thus bringing thefirst and second masses 2, 4 closer to one another.

Next (FIG. 4C), the force F_(X) increases progressively its value untilthe maximum value |A| is reached and with a direction such as to bringthe first and second masses 2, 4 closer to one another (e.g., F_(X)=−10pN). The force F_(Y) generates an oscillatory movement of the secondmass 4 around the first mass 2, which causes the donor molecule 6 andthe acceptor molecule 8 to approach each other. In the condition of FIG.4C, the donor molecule 6 and the acceptor molecule 8 are at a distancefrom one another such as to cause emission of light radiation.

In this context, it is emphasized that the condition of approach betweenthe donor molecule 6 and the acceptor molecule 8 such as to enableemission of light radiation is a function of the parameters of length ofthe strand 10 and of frequency of the electrical field applied.

In particular, light emission occurs if the frequency and amplitude ofthe electrical field applied are “tuned” with the length of the strand10 (a condition that is further referred to as “resonance”) for enablingcontact between the two masses 2 and 4, and thus between the donormolecule and the acceptor molecule; otherwise, there will not be lightemission. As used herein, “tuned” frequency and amplitude refers tovalues that cause the semicircular movement of the strand to terminatewith the donor molecule 6 and acceptor molecule 8 in contact, i.e., at adistance such as to enable energy exchange to occur, with lightemission. In the case where the semicircular movement terminates at agreater distance (i.e., with reference to the Cartesian system ofco-ordinates of FIG. 2, in the case where the mass 4 again assumes theco-ordinate y that is substantially 0 but, simultaneously, theco-ordinate x that is other than 0 for an amount greater than theeffective radius of the mass 2, and with any sign) exchange between thedonor molecule 6 and the acceptor molecule 8, and thus light emission,do not occur. In this case, the parameters are not “tuned”. The valuesto be set for the amplitude and frequency for them to be considered“tuned” depend upon the length of the strand considered. Based on thisis, in effect, the capacity of distinguishing between different lengthsand, in practice, of determining the sequence in question.

The effective length of the strand 10 is not known beforehand, andconsequently also the frequency (f) to be applied for reaching thecondition of light emission is not known beforehand. However, byfrequency sweeping in the range of frequencies already identifiedpreviously, it is possible to bring in tuned or resonance conditions, insequence, systems 1 having strands of various lengths. The interest, inanalyses of this type, is not in fact to know the absolute length of thestrand 10, but to acquire information useful for sequencing the strand10, and determining the relative lengths of strands 10 with differentterminations.

An estimate of the typical lengths may be obtained from the knowledge ofthe length of the gene amplified by the initial PCR step and from thelength of possible stretches of probe that could be used for binding themasses together. On the basis of one or both of these data, it ispossible to obtain beforehand a frequency range to be applied formovement of the mass 4.

FIG. 5 illustrates a portion of a device 18, obtained in MEMStechnology, comprising four wells 20, each of which is configured tocontain a liquid solution including a plurality of structures 1 of thetype described previously. The structures 1 in each well 20 includerespective strands 10. The second masses 4 in a same well 20 arefunctionalized to be able to couple to a same type of dideoxynucleotide(the same from among ddATP, ddTTP, ddGTP, and ddCTP). Second masses 4 indifferent wells are functionalized to be able to couple withdideoxynucleotides of types different from one another. It follows that,carrying out the frequency sweep on the four wells 20 of the device ofFIG. 5, for each value of resonance frequency (which corresponds to aspecific length of the strand 10) there will be prevalent light emissionfrom just one of said wells 20. Said light emission may thus be uniquelyassociated to one and only one dideoxynucleotide (one from among ddATP,ddTTP, ddGTP, and ddCTP) and, thus, to one and only one nucleotide base(a corresponding one from among A, T, G, and C).

The present applicant has estimated that the resonance frequencydecreases by approximately 75 MHz for each nucleotide base (assuming afrequency range of the order of gigahertz or fractions of a gigahertz,without prejudice to the aforementioned scalability of the quantities).This means that, for example, if a strand with a length of 40 basesresonates at 1 GHz, a strand with a length of 41 bases resonates at afrequency (f) of the AC field applied of approximately 1 GHz-75 MHz(i.e., 925 MHz), whereas a strand with a length of 39 bases resonates ata frequency f of the AC field applied of approximately 1 GHz+75 MHz. Itis in any case evident that the values provided above represent anembodiment and have been verified via simulations for strands withlengths comprised between 20 and 40 bases.

In general, noting that to given frequency values there is lightemission corresponding to a specific nucleotide base and knowing that,by increasing the length of the strand 10, the resonance frequencydecreases, it is possible to carry out a frequency sweep starting from amaximum frequency value and reduce said value either by predefined steps(for example, by steps of 20-50 MHz to be on the safe side) orcontinuously. By observing the sequence of “lighting” of the wells 20,the sequence of the bases that form the strand 10 under analysis isconsequently acquired.

Finally, with reference to FIG. 4D, by bringing, following a sinusoidalpattern, the value of the force F_(X) back to negative values (−10 pNaccording to the previous example), there is caused a progressiverecession of the second mass 4 from the first mass 2, and the system 1is brought back to the condition of FIG. 4A (time t=0).

The steps of FIGS. 4A-4E are carried out a plurality of times,continuously, for each frequency value (for example, maintaining a givenfrequency value for some microseconds, or some tens of microseconds).The cycle continues, as illustrated previously, by varying the value offrequency of the sinusoidal electrical field up to completion ofsequencing.

FIG. 6 is a schematic illustration of a microfluidic system 40 accordingto one aspect of the present disclosure. The microfluidic system 40comprises a microfluidic chip 32, including the device 18 of FIG. 5, andoptionally reserves of reagents for implementation of the Sanger method,according to embodiments of the present disclosure.

A PCR chip 33 has the function of carrying out amplification of the DNAsegments, according to the protocol envisaged by PCR, and introducingthe amplified segments into the microfluidic chip 32, for carrying outthe protocol of a Sanger type.

A generator of DC/AC electrical field 34 is operatively coupled to thedevice 18 for generation of the fields previously described, inparticular for generation of the forces F_(X) and F_(Y).

Finally, an optical reading unit 35 is operatively coupled to the device18 for optical reading of the signal of light radiation emitted duringthe use, as described previously.

The light information acquired by the optical reading unit 35 may besent, for an automatic analysis or an analysis assisted by an operator,to processing means, for example a computer (PC), 38.

Advantageously, the present disclosure provides a simple and low-costsystem for target sequencing of DNA subsequences of interest. The methoddescribed may be integrated with possible PCR steps already present inchips of a known type for carrying out a single analysis (in particular,in an automatic way) of the possible sequence identified by the PCR. Themethod according to the present disclosure is further simpler andeconomically advantageous as compared to global sequencing techniques ofa known type.

Finally, it is clear that modifications and variations may be made towhat has been described and illustrated herein, without therebydeparting from the scope of the present disclosure, as defined in theannexed claims.

For example, it is possible to implement the method described in amicrofluidic device provided with a single well, unlike what is shown inFIG. 5. In this case, each donor-acceptor pair is configured to emitlight radiation of a different color as a function of thedideoxynucleotide (ddATP, ddTTP, ddGTP, ddCTP) to which the mass 4 iscoupled. In this way, the sequence of the colors indicates the type ofbase sought for sequencing.

Further, according to a further embodiment, the AC field along X andalong Y may be generated using a signal other than a sinusoid, forexample an AC signal such as a square wave.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1. A method for sequencing a nucleic acid strand, comprising: providinga solution containing a plurality of truncated strands, extendingbetween a respective first end and a respective second end, and havinglengths different from one anther, wherein the second end terminateswith a respective dideoxynucleotide selected from the group consistingof ddATP, ddTTP, ddGTP, and ddCTP; functionalizing first masses by adonor molecule and second masses by an acceptor molecule, the donormolecule and the acceptor molecule being configured to emit lightradiation when they are arranged at a distance apart shorter than aminimum distance, and wherein each second mass has a mobility, in saidsolution, greater than the mobility of each first mass; coupling arespective first mass to the first end of each truncated strand;functionalizing the second masses so that each second mass may bind onlyto a respective terminal dideoxynucleotide of each strand from amongddATP, ddTTP, ddGTP, and ddCTP; coupling each second mass to theterminal dideoxynucleotide of each truncated strand; applying an ACelectrical field having frequency and amplitude to generate, on eachsecond mass, a net movement directed towards the first mass to which itis coupled via said truncated strand; varying said value of frequency ina predefined frequency range; acquiring light radiations emitted forrespective frequency values in said range; and associating each lightradiation acquired to a respective dideoxynucleotide and, thus, to arespective nucleotide base of the truncated strands.
 2. The methodaccording to claim 1, wherein functionalizing the second massescomprises creating a unique association between the color of the lightradiation emitted by a respective second mass and the respectivedideoxynucleotide to which the second mass is bound so that the color ofsaid emitted light radiation uniquely identifies a singledideoxynucleotide from among ddATP, ddTTP, ddGTP, and ddCTP.
 3. Themethod according to claim 1, wherein applying the AC electrical fieldcomprises applying an electrical field having a frequency and anamplitude to generate, on each second mass, a first force (F_(X))oriented along a first direction (X) and a second force (F_(Y)) orientedalong a second direction (Y), wherein the first direction corresponds tothe direction of extension of the truncated strand in a conditionthereof of maximum extension and the second direction is orthogonal tothe first direction.
 4. The method according to claim 3, furthercomprising, prior to the step of applying the AC electrical field,applying a DC electrical field having characteristics to generate, oneach second mass, a force to cause recession of the second mass from thefirst mass, thus causing said maximum extension of the truncated strandin said first direction.
 5. The method according to claim 1, wherein thefirst mass is a molecule with a high coefficient of hydrodynamicfriction.
 6. The method according to claim 1, wherein the first mass isa protein of a drag-tag type.
 7. The method according to claim 1,wherein the second mass has a net charge such that it is guided alongthe first and second directions under the action of the AC electricalfield.
 8. The method according to claim 1, wherein the first mass isfunctionalized with said donor molecule, and the second mass isfunctionalized with said acceptor molecule, said minimum distance beingless than 1 nm.
 9. The method according to claim 3, wherein the firstforce (F_(X)) and the second force (F_(Y)) are generated by quadraturesinusoidal signals with a frequency ratio equal to 2 or a multiple of 2,such that F_(X)=A·cos(ωt) and F_(Y)=B·sin(2ωt), where A and B are therespective values of signal amplitude, ω is the angular frequency, and tis the time variable.
 10. The method according to claim 9, wherein thefirst force (F_(X)) and the second force (F_(Y)) cooperate forgenerating a movement that displaces the second mass along asemi-curvilinear path around the first mass, imparting on the secondmass an oscillatory motion.
 11. The method according to claim 1, whereinthe step of varying the value of frequency in a predefined frequencyrange comprises carrying out a frequency sweep in said predefinedfrequency range for generating resonance conditions specific fordifferent lengths of the truncated strands.
 12. A device for sequencinga nucleic acid strand, comprising: a plurality of wells, wherein eachwell houses: a solution containing truncated strands extending between arespective first end and a respective second end and having lengthsdifferent from one another, wherein the second end terminates with arespective dideoxynucleotide from among ddATP, ddTTP, ddGTP, and ddCTP;first masses, coupled to the first end of each truncated strand, andfunctionalized with a donor molecule; second masses, functionalized forbinding only to a respective terminal dideoxynucleotide of the secondend of each strand, and functionalized with an acceptor molecule,wherein the donor molecule and the acceptor molecule are configured toemit light radiation when they are arranged at a distance apart shorterthan a minimum distance, an electrical-field generator configured togenerate an AC electrical field having a frequency and an amplitude toimpress, on each second mass, a net movement directed towards the firstmass to which each of the second masses is coupled via said truncatedstrand; control means for controlling the electrical-field generator,configured to vary the frequency value in a predefined frequency range;reading means, configured to acquire a plurality of light radiationsemitted by each well at respective frequency values in said range; andanalysis means, configured to associate each light radiation acquired toa respective dideoxynucleotide and, thus, to a respective nucleotidebase.
 13. The device according to claim 12, wherein the second massesare functionalized to create a unique association between the color ofthe light radiation emitted by a respective second mass and therespective dideoxynucleotide to which the second mass is bound so thatthe color of said light radiation emitted uniquely identifies a singledideoxynucleotide from among ddATP, ddTTP, ddGTP, and ddCTP.
 14. Thedevice according to claim 12, wherein the means for control of theelectrical-field generator are further configured to govern generationof an electrical field having a frequency and an amplitude to cause, oneach second mass, a first force (F_(X)) oriented along a first direction(X) and a second force (F_(Y)) oriented along a second direction (Y),wherein the first direction corresponds to the direction parallel to thetruncated strand in a condition of maximum extension thereof, and thesecond direction is orthogonal to the first direction.
 15. The deviceaccording to claim 14, wherein the means for control of theelectrical-field generator (34) are further configured to governgeneration of a DC electrical field to generate, on each second mass(4), a force that causes recession of the second mass (4) from the firstmass (2), bringing about said maximum extension of the truncated strand(10) in said first direction.
 16. The device according to claim 12,wherein the first mass is a molecule with high coefficient ofhydrodynamic friction.
 17. The device according to claim 12, wherein thefirst mass is a protein of a drag-tag type.
 18. The device according toclaim 12, wherein the second mass has a net charge such that it isguided along the first and second directions under the action of the ACelectrical field.
 19. The device according to claim 12, wherein thefirst mass is functionalized with said donor molecule, and the secondmass is functionalized with said acceptor molecule, said minimumdistance being less than 1 nm.
 20. The device according to claim 14,wherein the first force (F_(X)) and the second force (F_(Y)) aregenerated by quadrature sinusoidal signals with a frequency ratio equalto 2 or a multiple of 2, such that F_(X)=A·cos(ωt) and F_(Y)=B·sin(2ωt),where A and B are the respective values of signal amplitude, ω is theangular frequency, and t is the time variable.
 21. The device accordingto claim 20, wherein the first force (F_(X)) and the second force(F_(Y)) co-operate for generating a movement that displaces the secondmass along a semi-curvilinear path around the first mass, imparting uponthe second mass an oscillatory motion.
 22. The device according to claim12, wherein the means for control of the electrical-field generator arefurther configured to vary the frequency value in a predefined frequencyrange making a frequency sweep in said predefined frequency range forgenerating resonance conditions that are specific for different lengthsof the truncated strands.
 23. The device according to claim 12,comprising a microfluidic chip that houses in an integrated form: saidwells; one or more reserves of reagents for formation of said truncatedstrands, which may be fluidically coupled to the wells; and one or morechambers for carrying out a PCR, which may be fluidically coupled to thewells and/or to said one or more reserves of reagents.
 24. A device forsequencing nucleic acid strands, comprising: a plurality of wellsconfigured to receive aliquots of nucleic acid strands, one terminus ofeach nucleic acid strand being terminated by a respectivedideoxynucleotide selected from the group consisting of ddATP, ddTTP,ddGTP, and ddCTP, and each nucleic acid strand, at respective terminus,being functionalized with an energy donor and an energy acceptor capableof emitting light radiation when arranged at a distance below a minimumdistance; an electrical-field generator configured to generate an ACelectrical field of a frequency and amplitude in each well; controlmeans for controlling the electrical-field generator, configured to varythe frequency value in a predefined frequency range; and reading means,configured to acquire a plurality of light radiations emitted from eachwell at respective frequency values in said range.
 25. The deviceaccording to claim 24, further comprising analysis means configured toassociate each light radiation acquired to a respectivedideoxynucleotide and a respective nucleotide base.
 26. The deviceaccording to claim 24, wherein the control means is further configuredto govern generation of an electrical field having a frequency and anamplitude to cause a first force (F_(X)) oriented along a firstdirection (X) and a second force (F_(Y)) oriented along a seconddirection (Y), wherein the first direction is parallel to the nucleicacid strand in a condition of maximum extension thereof, and the seconddirection is orthogonal to the first direction.
 27. The device accordingto claim 26, wherein the first force (F_(X)) and the second force(F_(Y)) are generated by quadrature sinusoidal signals with a frequencyratio equal to 2 or a multiple of 2, such that F_(X)=A·cos(ωt) andF_(Y)=B·sin(2ωt), where A and B are the respective values of signalamplitude, ω is the angular frequency, and t is the time variable.